WO2016197443A1 - 3- 氨基 -2- 羟基 -4- 苯基 - 缬氨酰 - 异亮氨酸的新应用 - Google Patents

3- 氨基 -2- 羟基 -4- 苯基 - 缬氨酰 - 异亮氨酸的新应用 Download PDF

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WO2016197443A1
WO2016197443A1 PCT/CN2015/086003 CN2015086003W WO2016197443A1 WO 2016197443 A1 WO2016197443 A1 WO 2016197443A1 CN 2015086003 W CN2015086003 W CN 2015086003W WO 2016197443 A1 WO2016197443 A1 WO 2016197443A1
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hydroxy
isoleucine
phenyl
amino
inflammation
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戈梅
钱峰
饶敏
何慧琼
王涛
罗敏玉
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Shanghai Laiyi Center For Biopharmaceutical R & D
Shanghai Jiao Tong University
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Shanghai Laiyi Center For Biopharmaceutical R & D
Shanghai Jiao Tong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides

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  • the present invention belongs to the field of medicine, and in particular, relates to 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in preparation for prevention and treatment of inflammatory diseases and inflammation New applications in pharmaceutical preparations related to infectious diseases.
  • Inflammation is a very common and important basic pathological process. Any factor that can cause tissue damage, such as infection, non-infectious tissue damage (such as trauma, surgery, etc.) can cause inflammation, and These inflammations have many similar features (Barton GM. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest., 2008, 118: 413-420, for example, inflammation caused by bacterial infection, bacterial secretion Endotoxin (lipopolysaccharides, abbreviated as LPS) is a major cause of bacterial pathogenesis and can cause a series of inflammatory reactions in the body. LPS-induced inflammatory signals are associated with Toll-like receptors (TLRs), etc.
  • TLRs Toll-like receptors
  • LPS acts on TLRs receptors on the cell membrane, and the expression of cascaded genes changes through intracellular signaling.
  • LPS can stimulate the synthesis and release of inflammatory factors (such as NO, TNF- ⁇ , IL-6, IL- ⁇ , etc., causes systemic inflammatory reactions, causing toxic shock, systemic inflammatory response syndrome (SIRS) and Inflammatory diseases such as multiple organ dysfunction syndrome (MODS) (Wang Xiaodong, Endotoxin Neutralizing Protein and Its Role in Prevention and Treatment of Sepsis[J], Foreign Medical Physiology, Pathology and Clinical Section, 2001, 21(2) : 14 4-146; Li Ying, Wang Xingpeng, Research progress in blocking endotoxin signaling pathway in the treatment of sepsis or septic shock [J], Chinese Journal of Emergency Medicine, 2003, 12(2): 135-137; Ying, Guo Zaichen, Treatment of septic shock [J], Chinese Journal of Practical Pediatrics, 2007, 22
  • Ubenimex also known as Bestatin
  • Bestatin is a common anti-tumor adjuvant that enhances immunity. Function, for anticancer chemotherapy, adjuvant therapy for radiotherapy, etc.
  • Bestatin an inhibitor of aminopeptidase B, produced by actinomycetes, (29). Pp. 97-99; Muskardin, DT, Voelkel. NF & Fitzpatrick, FA (1994). Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase. (48).
  • 3-Amino-2-hydroxy-4-phenyl-prolyl-isoleucine is a novel derivative similar in structure to Bestatin, and the applicant of the present invention is in the application number 201110076554.6
  • the Chinese invention patent discloses 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, which has the chemical structural formula:
  • the inventors of the present invention have further studied the function of the compound 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, and found that the compound has obvious effects on acute inflammation caused by LPS. Inhibition. Accordingly, it is an object of the present invention to provide a 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in the preparation of an inflammatory inhibitor or a medicament for the prevention and treatment of an inflammatory disease. New applications in formulations. Problem solution
  • a first aspect of the present invention provides 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in the preparation of a medicament for the prevention and treatment of inflammatory diseases and inflammation
  • the chemical structural formula of the 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine is:
  • the inflammatory disease and infectious diseases associated with inflammation include bacterial infection, uncomplicated cystitis, bronchitis, trauma, postoperative inflammatory reaction, accidental injury, myocardial infarction, tuberculosis or sarcoidosis, Sepsis, metastatic tumors, active rheumatoid arthritis, seronegative spinal arthritis, immune vasculitis, rheumatic polymyopathy, Crohn's disease, inflammation of deep vein thrombosis, etc.
  • the inventors administered a bacterial endotoxin LPS-induced acute lung injury model in mice, intraperitoneal administration of 3-amino- 2-hydroxy-4-phenyl-prolyl-isoleucine drug, detecting the effect of drugs on acute lung injury caused by LPS, and testing for 3-amino-2-hydroxy-4-phenyl-prolyl-
  • the anti-inflammatory effect of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine was also evaluated, and the inventors selected the mouse mononuclear-macrophage cell line Raw264.7 for testing.
  • the expression and release of inflammatory factor NO induced by LPS were detected, and it was found that the release of inflammatory factor NO induced by LPS was greatly reduced.
  • a second aspect of the present invention provides a pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, comprising a therapeutically effective amount of the active ingredient and a pharmaceutically acceptable carrier or excipient
  • the active ingredient is 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, an optical isomer thereof or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (such as a lyophilized powder injection).
  • Pharmaceutically acceptable salts can be pharmacologically and pharmaceutically acceptable.
  • the pharmacologically and pharmaceutically acceptable salt thereof may be an alkali metal or alkaline earth metal salt, preferably a sodium salt, a potassium salt, a magnesium salt or a calcium salt.
  • the pharmaceutical preparations involve enteral (e.g., oral, sublingual or rectal administration), parenteral or topical (e.g., transdermal pharmaceutical formulations) dosage forms.
  • An organic or inorganic substance which does not react with the active ingredient may be used as a carrier such as water, oil, benzyl alcohol, polyethylene glycol, triacetin or other fatty acid glycerides, gelatin, lecithin, cyclodextrin, lactose or A sugar such as starch, magnesium stearate, talc or cellulose.
  • the oral administration is preferably a tablet, a dragee, a capsule, a powder, a syrup, a concentrate or a drop, a suppository for rectal administration, an aqueous solution or an oil solution for parenteral administration, or a lyophilizate.
  • Suspensions, emulsions or implants may also be employed, and patches or creams may be used for topical administration.
  • Pharmaceutical preparations for parenteral administration comprise sterile aqueous or anhydrous injections of the active compound, preferably a solution which is isotonic with the blood of the recipient.
  • These pharmaceutical preparations may contain stabilizers, additives to control the release of pharmaceutically active compounds, antioxidants, buffers, bacteriostats, and adjuvants for the preparation of isotonic solutions.
  • Aqueous and anhydrous sterile suspensions may contain There are suspension additives and thickeners.
  • the pharmaceutical preparations may be dispensed in single or multi-dose containers, such as sealant bottles, or as lyophilized preparations, if desired, using sterile liquids such as water or saline solutions. Sterile powders, granules or tablets can also be used in the same manner.
  • the pharmaceutical preparation can be used for the prevention and treatment of inflammation and inflammation-related diseases in humans and animals.
  • MPO myeloperoxidase
  • FIG. 2A and 2B are graphs showing the results of detection of total protein content in lung lavage fluid in a model of LPS-induced acute lung injury in mice; wherein, FIG. 2A is the total amount of pulmonary lavage fluid after 4 hours of administration of LPS. Protein concentration determination, Figure 2B is the determination of the total protein concentration of the lung lavage fluid after 12 hours of LPS administration.
  • Figure 3 shows the presence or absence of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine (LYR) after 4 hours of LPS induction.
  • LYR 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine
  • FIG. 4 is a graph showing the results of immunoblotting of lNOS-induced expression of iNOS and COX-2 protein in LPS-induced lung tissue by 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine (LYRM03).
  • 5A is a graph showing the results of two immunoblotting results of iNOS protein expression in LPS-induced Raw264.7 cells.
  • Figure 5B is a quantitative analysis chart of iNOS protein expression in LPS-induced Raw264.7 cells
  • FIG. 6 is a graph showing the detection results of the NO release amount in the culture supernatant by the Griess method.
  • the present invention provides a pharmaceutical preparation for preparing an infectious disease for preventing and treating inflammatory diseases and inflammation, as an active ingredient of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine.
  • the inventors numbered 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as LYRM03.
  • the specific operation of the detection method used in the following embodiments is as follows:
  • C57/BL mice were grouped into groups of at least 5, weighed, and administered intratracheally with saline (negative control group) and 5 mg/kg LPS (inflammation group), respectively, before LPS injection.
  • saline negative control group
  • LPS inflammation group
  • Xiaoyan 10 minutes later, and 12 hours later, intraperitoneal injection of Bestatin (except for the negative control group), 0, 4, 24 hours later, the heart was taken, and the lungs were repeatedly washed with 1 ml of PBS.
  • the lung tissue was collected after collecting the lavage solution, the total protein content in the lung lavage fluid was detected, the activity of myeloperoxidase (MPO) in the lung tissue was detected, and the tissue sections were prepared, and the cytokines and inflammation in the lung tissue were detected by real-time PCR.
  • the expression of factors mainly TNF-ot, IL-6, IL- ⁇ , IL-12, etc.).
  • Trizol lysed cells extracted RNA, and reverse-transcribed PCR to obtain cDNA, which was used as a template for real-time PCR to examine DNA transcription levels of inflammation-related cytokines and inflammatory molecules induced before and after drug action.
  • NO easily forms NO 2 - in an aqueous solution, and under acidic conditions, NO 2 - reacts with a diazonium salt sulfonamide to form a diazo compound, and further undergoes a coupling reaction with naphthyl vinyl diamine.
  • the product concentration has a linear relationship with the N 0 2 concentration and has a maximum absorption peak at 540-560 nm. Using this principle, the amount of NO released from the cell supernatant can be detected.
  • WM 3- 3 ⁇ 4-2-hydroxy 3 ⁇ 4-4-benzene 3 ⁇ 4 -decanoyl-iso-t-acid reduces LPS induction to the market ⁇ ⁇ ⁇ ⁇ ⁇ :
  • LPS inflammation group tracheal injection of 5 mg / kg LPS 50 ⁇ 1;
  • LPS+LYRM03 treatment group LYRM03 (10 mg/kg each time, about 200 ⁇ l) was intraperitoneally injected 12 hours before and 10 minutes after intratracheal injection of LPS (5 mg/kg, about 50 ⁇ l);
  • MPO myeloperoxidase
  • FIG. 2A and FIG. 2B are graphs showing the results of detection of total protein content in lung lavage fluid in a model of LPS-induced acute lung injury in mice; wherein, FIG. 2A is the total amount of pulmonary lavage fluid after 4 hours of administration of LPS. Protein concentration determination, Figure 2B is the determination of the total protein concentration of the lung lavage fluid after 12 hours of LPS administration.
  • Example 3 After inducing 4 hours of LPS (5 mg/kg), the right lobe lung tissue of the mouse was taken, and in the presence or absence of LYRM03, the cells in the lung tissue of the mouse were detected by real-time PCR. A graph of the expression levels of factors and inflammatory factors, the results are shown in Figure 3.
  • the method of administration was the same as that described in Example 1. 4 hours and 24 hours after LPS (5mg/kg) induction, the largest lung lobe on the left side of the mouse was taken, lysed after homogenization, total protein concentration was determined, and iNOS and COX-2 were detected by western blot. Protein expression, actin was loaded and the results are shown in Figure 4.
  • LYRM03 can alleviate the acute inflammatory injury response of bacterial endotoxin LPS, including neutrophil infiltration and expression of inflammatory molecules, indicating that LYRM03 has an inflammatory therapeutic effect.
  • Example 3 3- 3 ⁇ 4-2-hydroxy 3 ⁇ 4-4-benzene 3 ⁇ 4-quinoyl-iso-acid (LYRM03) anti-inflammatory effect 3 ⁇ 4 :
  • This example uses the Raw264.7 mouse macrophage cell line as an example to compare the effect of LYRM03 on the expression of inflammatory factor NO induced by LPS, including the expression of iNOS protein in cells after 24 hours of LPS stimulation (Fig. 5A and Figure 5B) and the amount of NO released in the cell culture supernatant (Figure 6).
  • Raw264.7 cells were seeded in a 12-well plate at a density of 3.5 ⁇ 10 5 /well, cultured overnight with 1640 medium containing 10% fetal bovine serum, and LYRM03 was incubated for 30 minutes at a concentration of 0.2 mg/ml. An equal volume of medium was induced by stimulation with Olg/ml of LPS for 24 hours. The supernatant was collected and lysed for immunoblot analysis.
  • Figure 5A shows the results of two immunoblots of iNOS protein expression, ⁇ -actin as a control
  • Figure 5B shows the quantitative analysis of iNOS protein expression, the optical density ratio of iNOS and ⁇ -a C ti n using Im a g e J software Calculate, take the results of three independent experiments to calculate the standard error of the mean, compare the LYRM03 results with the L PS group, and make a significant difference analysis, where ** ⁇ ⁇ 0.01.
  • the Griess method was used to detect the amount of NO released from the culture supernatant.
  • the cell culture and stimulation induction were as described in Fig. 5A and Fig. 5B, and the culture supernatant was taken, and the amount of NO released therein was detected by a kit.
  • the mean standard error was calculated from the results of three independent experiments.
  • the LYRM03 results were compared with the LPS group for significant difference analysis, ⁇ 0.05, * ⁇ 0.01.
  • the release of NO has a reduction of more than 50%.
  • the present invention provides a novel use of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, which is prepared as an active ingredient for the prevention and treatment of inflammatory diseases and Use in pharmaceutical preparations for inflammation-related infectious diseases.
  • the present invention also provides a pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, said activity
  • the component is 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, an optical isomer thereof or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (e.g., a lyophilized powder injection).

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Abstract

一种3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸的新应用，即其作为活性成分在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用，以及一种用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂，含有治疗有效量的活性成分和药学上可接受的载体或赋形剂，所述活性成分为3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸、其旋光异构体或其药物学上可接受的盐。

Description

3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸的新应用 技术领域
[0001] 本发明属于医药领域， 具体地说， 涉及 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸 作为活性成分在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物 制剂中的新应用。
背景技术
[0002] 炎症是一种十分常见而又重要的基本病理过程， 任何能够弓 I起组织损伤的因素 ， 如感染、 非感染性组织损伤 （如创伤、 手术等） 均可导致炎症的发生， 且这 些炎症具有很多相似的特征 (Barton GM. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest., 2008, 118: 413-420 以细菌感染导致的炎症为例， 细菌分泌的内毒素 （细菌脂多糖 lipopolysaccharides ，简称 LPS) 是细菌致病的主要因素， 可引起机体的一系列炎症反应。 LPS诱导的 炎症信号与 Toll样受体 （TLRs) 家族等有关。 当机体受到细菌感染吋， LPS作用 于细胞膜上 TLRs受体， 通过胞内信号传递， 级联基因表达发生变化， LPS可刺 激体内多种细胞因子合成和释放炎性因子 （如 NO， TNF-α, IL-6, IL-Ιβ等） ， 导致全身性炎症反应发生， 引起中毒性休克、 全身炎症反应综合征 （SIRS) 和 多器官功能障碍综合征 （MODS) 等炎症疾病 （王晓东， 内毒素中和蛋白及其在 脓毒症防治中的作用 [J]， 国外医学生理、 病理科学与临床分册， 2001,21(2): 14 4-146； 丽颖、 王兴鹏， 阻断内毒素信号传导通路治疗脓毒症或脓毒性休克的研 究进展 [J]， 中华急诊医学杂志， 2003， 12(2)： 135-137； 王颖、 郭在晨， 感染性 休克的治疗 [J]， 中华实用儿科临床杂志， 2007， 22(6)： 403-405； 张雪梅、 熊焕 章， LPS诱导的炎症反应信号传导通路研究进展， 中国兽医杂志， 2010， 46(7)： 45-47) 。 因此， LPS可视为细菌的主要致病因子， 在细菌感染的发病机理中起 着十分重要的作用。 同样， 在组织损伤吋， 机体会分泌大量细胞因子以修复损 伤， 但当反应过度吋， 也会引发 SIRS、 MODS等炎症疾病。
[0003] 乌苯美司 （Ubenimex, 又名 Bestatin) 是常见的抗肿瘤辅助药物， 可增强免疫 功能， 用于抗癌化疗、 放疗的辅助治疗等 （Umezawa, H.， Aoyagi, T.， Suda, H.,
Hamada, M. & Takeuchi, T. (1976). Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes, (29). Pp. 97-99; Muskardin, D.T., Voelkel. N.F. & Fitzpatrick, F.A. (1994). Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase. (48).
pp.131-137; Hirayama, Y; Sakamaki, S; Takayanagi, N; Tsuji, Y; Sagawa, T; Chiba, H; Matsunaga, T; Niitsu, Y. (2003). "Chemotherapy with ubenimex corresponding to patient age and organ disorder for 18 cases of acute myelogeneous leukemia in elderly atients― effects, complications and long-term survival". Gan to kagakuryoho. Cancer
Figure imgf000004_0001
[0005] 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸是与乌苯美司 （Bestatin) 结构相似的新 型衍生物， 本发明的申请人在申请号为 201110076554.6的中国发明专利中首次公 幵该化合物 3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸， 其化学结构式为：
[0006]
Figure imgf000004_0002
[0007] 在申请号为 201110076555.0的中国发明专利中进一步公幵了化合物 3-氨基 -2-羟 基 -4-苯基 -缬氨酰-异亮氨酸作为氨肽酶抑制剂的应用， 其活性优于乌苯美司 （Be statin) ， 可用作肿瘤治疗或辅助治疗药物， 具有更好的体内免疫增强作用， 如 能增强 T细胞和杀伤性 NK细胞活性和功能等， 但对在如细菌引起的急性炎症中 的作用尚未见报道。
技术问题
[0008] 本发明的发明人在对化合物 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸的功能做进 一步研究中发现， 该化合物对于 LPS引起的急性炎症具有明显的抑制作用。 因此 ， 本发明的目的在于提供一种 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸作为活性成 分在制备炎症抑制剂或制备用于预防和治疗炎症疾病的药物制剂中的新应用。 问题的解决方案
技术解决方案
[0009] 为实现上述目的， 本发明采用以下技术方案：
[0010] 本发明的第一个方面是提供 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸作为活性成 分在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应 用， 所述 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸的化学结构式为：
Figure imgf000005_0001
[0012] 其中， 所述炎症疾病和与炎症相关的感染疾病包括细菌感染、 无并发症的膀胱 炎、 支气管炎、 外伤、 术后炎症反应、 意外伤害、 心肌梗塞、 结核病或肉状瘤 病、 脓毒症、 转移性肿瘤、 活动期类风湿性关节炎、 血清阴性的脊椎关节炎、 免疫性血管炎、 风湿性多肌病、 局限性回肠炎、 深部静脉血栓形成的炎症等。
[0013] 发明人通过构建细菌内毒素 LPS诱导的小鼠急性肺损伤模型， 腹腔给予 3-氨基- 2-羟基 -4-苯基 -缬氨酰-异亮氨酸药物， 检测药物对 LPS引起的急性肺损伤的作用 ， 并检验 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸对细胞因子、 炎症分子等表达的 影响。 同吋， 还评价了 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸的抗炎效果， 发明 人选择小鼠单核 -巨噬细胞系 Raw264.7进行测试， 检测 LPS诱导的炎性因子 NO表 达和释放， 检测后发现， LPS诱导的炎性因子 NO的释放量大幅减少。
[0014] 还通过髓过氧化物酶 （Myeloperoxidase, ΜΡθ) 活性检测、 肺盥洗液中总蛋白 含量检测和 HE染色组织切片， 观测并检验嗜中性粒细胞的浸润情况， 以评价 LP S诱导的急性肺损伤严重程度。 检测结果表明， 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异 亮氨酸对 LPS诱导的小鼠肺部嗜中性粒细胞浸润有抑制作用。
[0015] 本发明的第二个方面是提供一种用于预防和治疗炎症疾病和与炎症相关的感染 疾病的药物制剂， 含有治疗有效量的活性成分和药学上可接受的载体或赋形剂 ， 所述活性成分为 3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸、 其旋光异构体或其药 物学上可接受的盐。
[0016] 优选地， 所述药物制剂的剂型包括片剂、 胶囊剂、 口服液、 颗粒剂或注射剂 （ 如冻干粉针剂） 。
[0017] 药物学上可接受的盐可以是药理学上和其药物学上都接受的。 药理学上和其药 物学上可接受的盐可以是碱金属盐或碱土金属盐， 优选钠盐、 钾盐、 镁盐或钙
[0018] 所述药物制剂涉及经肠的 （例如口服、 舌下含服或直肠给药） 、 胃肠外的或局 部的 （如透皮药物制剂） 剂型。 不与活性成分反应的有机或无机物可以作为载 体， 如水、 油、 苯甲醇、 聚乙二醇、 甘油三乙酸酯或其它脂肪酸甘油酯、 明胶 、 卵磷脂、 环糊精、 乳二糖或淀粉等糖类、 硬脂酸镁、 滑石或纤维素。 口服用 药优选片剂、 糖衣丸剂、 胶囊、 粉、 糖浆、 浓缩剂或滴剂， 直肠给药优选栓剂 ， 胃肠外给药优选水溶液或油溶液， 或冻干剂。 也可以使用混悬剂、 乳剂或植 入剂， 局部用药可以用贴剂或乳膏剂。 胃肠外使用的药物制剂包括活性化合物 的无菌含水或无水注射液， 在此优选与受体血液等渗的溶液。
[0019] 这些药物制剂可以含有稳定剂、 控制药学活性化合物释放的添加剂、 抗氧剂、 缓冲剂、 抑菌剂和用于制备等渗溶液的佐剂。 含水和无水的无菌混悬剂可以含 有混悬添加剂和增稠剂。 药物制剂可以分装在单剂量或多剂量容器如密封剂瓶 中， 也可以作为经冻干的制品保存， 如需要， 使用吋可用无菌液体如水或盐溶 液配制。 还可以以同样的方式使用无菌粉剂、 颗粒剂或片剂。
[0020] 所述药物制剂可用于人和动物中预防和治疗炎症和炎症相关疾病。
发明的有益效果
对附图的简要说明
附图说明
[0021] 图 1为构建的 LPS诱导的小鼠急性肺损伤模型中， 髓过氧化物酶 （MPO) 活性 检测结果图表。
[0022] 图 2A和图 2B为构建的 LPS诱导的小鼠急性肺损伤模型中， 肺盥洗液中总蛋白含 量检测结果图表； 其中， 图 2A为 LPS给药 4小吋后肺盥洗液的总蛋白浓度测定， 图 2B为 LPS给药 12小吋后肺盥洗液的总蛋白浓度测定。
[0023] 图 3为 LPS诱导 4小吋后， 在有无 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸 （LYR
M03) 的情况下， 小鼠肺组织中细胞因子和炎症因子的表达水平图表。
[0024] 图 4为 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸 （LYRM03) 对 LPS诱导的肺组织 中 iNOS和 COX-2蛋白表达的免疫印迹结果图表。
[0025] 图 5A为 LPS诱导的 Raw264.7细胞中， iNOS蛋白表达的两次免疫印迹结果图表
[0026] 图 5B为 LPS诱导的 Raw264.7细胞中， iNOS蛋白表达的量化分析图表；
[0027] 图 6为 Griess法检测培养上清中 NO释放量的检测结果图表。
本发明的实施方式
[0028] 下面通过具体实施例对本发明进行详细和具体的介绍， 以使更好的理解本发明 ， 但是下述实施例并不限制本发明范围。
[0029] 本发明提供了 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸作为活性成分在制备用于 预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用。 在本实施 例中， 发明人将 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸编号为 LYRM03。 [0030] 以下实施例中所使用的检测方法的具体操作如下：
[0031] 1、 构建 LPS诱导的急性炎症模型：
[0032] 将 C57/BL小鼠分组， 每组至少 5只， 称重， 分别用生理盐水 （阴性对照组） 和 5 mg/kg的 LPS (炎症组） 进行气管给药， 在 LPS注射前 12小吋， 10分钟后以及 12 小吋后给予乌苯美司 （Bestatin) 腹腔注射 （阴性对照组除外） ， 0、 4、 24小吋 后处死， 心脏取血， 用 lml的 PBS反复冲洗肺部， 收集盥洗液后取肺组织， 检测 肺盥洗液中总蛋白的含量， 进行肺组织髓过氧化物酶 （MPO) 活性的检测， 并 制备组织切片， 荧光定量 PCR检测肺组织中细胞因子和炎症因子的表达情况 （主 要是 TNF-ot， IL-6, IL-Ιβ, IL-12等） 。
[0033] 2、 荧光定量 PCR (QPCR) ：
[0034] Trizol裂解细胞， 抽提 RNA， 逆转录 PCR获得 cDNA， 以此为模板进行荧光定量 PCR, 检验药物作用前后诱导的炎症相关细胞因子和炎性分子等的 DNA转录水 平变化。
[0035] 3、 免疫印迹 （Western Blot) ：
[0036] 裂解并收集细胞， 变性处理， SDS电泳 80V 2小吋， 转膜 80V 1小吋， 5%牛奶封 闭半小吋， 4°C孵育一抗过夜， TBST缓冲液 (Tris-Buffered Saline and Tween 20 ) 洗膜 3次， 常温孵育二抗 1小吋， TBST洗膜 3次， Odyssey远红外观测仪上观察 药物作用前后诱导的一氧化氮合成酶 iNOS蛋白表达水平变化等。
[0037] 4、 Griess法检测 NO释放量：
[0038] NO在水溶液中极易形成 NO 2-， 在酸性条件下， NO 2 -与重氮盐磺胺发生重氮反 应， 生成重氮化合物， 进一步与萘基乙烯基二胺发生偶合反应， 其产物浓度与 N 0 2浓度具有线性关系， 在 540-560nm处有最大吸收峰。 利用此原理可检测细胞 上清中 NO的释放量。
[0039] WM 3- ¾-2-羟¾-4-苯¾ -缬 酰-异 t酸 (LYRM03) 降低 LPS诱导导 至删市咅 耆 瞧田隱闰：
[0040] 将 C57/BL小鼠 30只， 随机分成 3组， 每组 10只， 称重， 分别进行如下操作： [0041] 阴性对照组： 气管注射 0.9%的生理盐水 50μ1;
[0042] LPS炎症组： 气管注射 5mg/kg的 LPS 50μ1; [0043] LPS+LYRM03治疗组： 在气管注射 LPS (5mg/kg， 约 50μ1) 前 12小吋和 10分钟 后， 两次腹腔注射 LYRM03 (每次 10mg/kg， 约 200μ1) ；
[0044] 0、 4、 24小吋后处死， 心脏取血， 同吋取小鼠肺组织， 分别进行肺组织髓过氧 化物酶 （ΜΡΟ) 活性的检测和肺盥洗液中总蛋白的含量检测， 检测结果分别如 图 1、 图 2Α、 图 2Β和图 3所示。
[0045] 通过上述三种检测来检验嗜中性粒细胞的浸润情况， 以评价 LPS诱导的急性肺 损伤严重程度。
[0046] 图 1为构建的 LPS诱导的小鼠急性肺损伤模型中， 髓过氧化物酶 （MPO) 活性 检测结果图表。
[0047] 从图 1的检测结果显示， LPS (5mg/kg) 气管给药 4小吋和 24小吋后， 与生理盐 水对照组 （Saline) 相比， 小鼠肺部 MPO活性均有 3倍左右的增加， 而在 LYRM0 3 ( I0mg/kg) 干预下， MPO活性增加不超过 2倍 （其中， ** Ρ<0.01， *** Ρ
<0.005，
Figure imgf000009_0001
。
[0048] 图 2Α和图 2Β为构建的 LPS诱导的小鼠急性肺损伤模型中， 肺盥洗液中总蛋白含 量检测结果图表； 其中， 图 2A为 LPS给药 4小吋后肺盥洗液的总蛋白浓度测定， 图 2B为 LPS给药 12小吋后肺盥洗液的总蛋白浓度测定。
[0049] 从图 2A和图 2B的肺盥洗液中总蛋白的含量测定结果显示， LYRM03 ( lOmg/kg
) 对 LPS诱导的肺部总蛋白浓度增加有约 50% (图 2A: 4小吋） 和接近 100% (图
2B : 24小吋） 的抑制。
[0050] 实施例 2、 3- ¾-2-羟¾-4-苯¾ -缬 酰-异 t酸 (LYRM03) 抑制 LPS诱导的 细朐因子、 炎症分子表 i大：
[0051] 参照实施例 1的方法， 在 LPS (5mg/kg) 诱导 4小吋后， 取小鼠右叶肺组织， 在 有无 LYRM03的情况下， 进行荧光定量 PCR检测小鼠肺组织中细胞因子和炎症因 子的表达水平图表， 结果如图 3所示。
[0052] 给药方法与实施例 1中所述方法一样。 分别在 LPS (5mg/kg) 诱导后 4小吋和 24 小吋， 取小鼠左侧最大肺叶， 匀浆后裂解， 测定总蛋白浓度， 免疫印迹 （wester n blot) 检测 iNOS和 COX-2的蛋白表达， actin做上样对照， 结果如图 4所示。
[0053] 从图 3的细胞因子和炎症因子表达水平检测结果显示， 经 LPS刺激后 3-6小吋， 小鼠肺部和血液中的细胞因子、 炎症因子 （如 TNF-a， IL-6, IL-Ιβ, 环氧酶 CO X-2等） 会有一个明显的表达激增， 通过荧光定量 PCR检测发现， LYRM03的施 加可有效抑制小鼠肺组织中这些炎症因子的表达。 例如， 在 LPS刺激前 12小吋和 10分钟后注射 LYRM03 (I0mg/kg) ， 小鼠肺组织中的 TNF-α表达量是 LPS组的 3 1.3%, IL-Ιβ的表达量是 LPS组的 36.8<¾， 环氧酶 COX-2的表达量是 LPS组的 59% ， 其它细胞因子的表达量也有明显降低。
[0054] 图 4的免疫印迹 （western blot) 检测结果显示， 小鼠肺组织中的 NO合成酶 iNOS 和 COX-2蛋白表达量在 LYRM03的影响下有所下降。
[0055] 上述结果表明， LYRM03可以减轻细菌内毒素 LPS弓 |起的急性炎症损伤反应， 包括嗜中性粒细胞的浸润和炎症分子的表达， 显示了 LYRM03具有炎症治疗作用
[0056] 实施例 3、 3- ¾-2-羟¾-4-苯¾ -缬 酰-异 t酸 （LYRM03) 的抗炎效 ¾:
[0057] 本实施例以 Raw264.7小鼠巨噬细胞系为例， 比较 LYRM03对 LPS诱导的炎性因 子 NO表达的影响， 包括 LPS刺激 24小吋后细胞中 iNOS蛋白的表达 （图 5A和图 5B ) 和细胞培养上清中 NO的释放量 （图 6) 。
[0058] 如图 5A和图 5B所示为 LPS诱导的 Raw264.7细胞中 iNOS的蛋白表达水平检测。
将 Raw264.7细胞以 3.5X10 5/孔的密度接种于 12孔板中， 用含 10%胎牛血清的 1640 培养液培养过夜， LYRM03以 0.2mg/ml的浓度提前孵育 30分钟， 对照组为等体积 培养液 （medium) ， 用 O.l g/ml的 LPS刺激诱导 24小吋。 收集上清， 裂解细胞进 行免疫印迹分析。 其中， 图 5A为 iNOS蛋白表达的两次免疫印迹结果， β-actin为 对照； 图 5B为 iNOS蛋白表达的量化分析， iNOS和β-aCtin的光密度比值用Image J 软件测算， 取三次独立实验的结果计算平均值标准误差， 比较 LYRM03结果与 L PS组， 进行显著性差异分析， 其中 ** Ρ<0.01。
[0059] 如图 6所示为 Griess法检测培养上清中 NO释放量。 细胞培养和刺激诱导如图 5A 和图 5B所述， 取培养上清， 试剂盒检测其中 NO的释放量。 取三次独立实验的结 果计算平均值标准误差， 比较 LYRM03结果与 LPS组， 进行显著性差异分析， 其 中 <0.05， * <0.01。
[0060] 由图 5A、 图 5B、 图 6检测结果看出， 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸 （ LYRM03) 对 LPS诱导的 Raw264.7细胞中的蛋白表达水平和 LPS诱导的炎性因子
NO的释放量均有超过 50%的减少。
[0061] 因此， 本发明提供了 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸的新应用， 即其作 为活性成分在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制 剂中的应用。 由此， 本发明还提供了一种用于预防和治疗炎症疾病和与炎症相 关的感染疾病的药物制剂， 含有治疗有效量的活性成分和药学上可接受的载体 或赋形剂， 所述活性成分为 3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸、 其旋光异构 体或其药物学上可接受的盐。 进一步优选地， 优选地， 所述药物制剂的剂型包 括片剂、 胶囊剂、 口服液、 颗粒剂或注射剂 （如冻干粉针剂） 。
[0062] 以上对本发明的具体实施例进行了详细描述， 但其只是作为范例， 本发明并不 限制于以上描述的具体实施例。 对于本领域技术人员而言， 任何对本发明进行 的等同修改和替代也都在本发明的范畴之中。 因此， 在不脱离本发明的精神和 范围下所作的均等变换和修改， 都应涵盖在本发明的范围内。

Claims

权利要求书
[权利要求 1] 3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸作为活性成分在制备用于预防 和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用， 所述 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸的化学结构式为：
Figure imgf000012_0001
[权利要求 2] 根据权利要求 1所述的应用， 其特征在于， 所述炎症疾病和与炎症相 关的感染疾病包括细菌感染、 无并发症的膀胱炎、 支气管炎、 外伤、 术后炎症反应、 意外伤害、 心肌梗塞、 结核病或肉状瘤病、 脓毒症、 转移性肿瘤、 活动期类风湿性关节炎、 血清阴性的脊椎关节炎、 免疫 性血管炎、 风湿性多肌病、 局限性回肠炎、 深部静脉血栓形成的炎症
[权利要求 3] —种用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂， 其特征在于， 含有治疗有效量的活性成分和药学上可接受的载体或赋 形剂， 所述活性成分为 3-氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸、 其旋 光异构体或其药物学上可接受的盐。
[权利要求 4] 根据权利要求 3所述的药物制剂， 其特征在于， 所述药物制剂的剂型 包括片剂、 胶囊剂、 口服液、 颗粒剂或注射剂。
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